A pharmaceutical composition for treating acetaminophen-induced liver injury and use thereof

Abstract

The application discloses a kind of pharmaceutical composition for treating liver injury of acetylaminophenol permeation and application, belong to biological medicine field.The composition is composed of N-acetylcysteine or its pharmaceutically acceptable salt, 8-oxo guanine DNA glycosylase 1 specific activator, preferably the activator is TH10785 and its derivative, synergistic effect of both.The composition can break through the traditional 8 hours treatment window of NAC, realize late intervention in poisoning, play the efficacy by repairing DNA oxidative damage, inhibiting Z-DNA generation, blocking ZBP1 path, inhibiting hepatocyte pyroptosis and liver inflammation.Drug can be made into various dosage forms, and matched special treatment kit.The application prolongs clinical treatment time, and double target points synergistically improve treatment effect, target point is novel, applicability is strong, with good clinical transformation prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a pharmaceutical composition for treating acetaminophen-induced liver injury and its application. Background Technology

[0002] Acetaminophen (APAP) is one of the most widely used antipyretic and analgesic drugs in clinical practice worldwide. It has a good safety profile at conventional therapeutic doses, but overdose is a core cause of acute liver injury and acute liver failure. It is also the leading cause of death in clinical liver transplantation and acute liver failure in Western countries, posing a significant clinical hazard.

[0003] Acetaminophen-induced acute liver injury (AILI) has a well-defined pathological mechanism: excessive APAP intake is metabolized by the hepatic CYP2E1 enzyme to produce the highly active and toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI). Under normal physiological conditions, the glutathione (GSH) stored in the liver can neutralize NAPQI and relieve drug toxicity; however, when APAP is excessive, the large amount of NAPQI generated will rapidly deplete the liver's GSH reserves. The remaining unneutralized NAPQI can covalently bind to mitochondrial proteins in hepatocytes, inducing mitochondrial dysfunction and severe oxidative stress, ultimately leading to massive hepatocyte death, central lobular necrosis, and irreversible acute liver injury.

[0004] Currently, the only standard-acting drug for the clinical treatment of AILI is N-acetylcysteine ​​(NAC). Its mechanism of action is to replenish GSH levels in hepatocytes, neutralize early-stage NAPQI, and block the progression of toxic damage. However, NAC has unavoidable clinical technical limitations: its effective treatment window is extremely narrow, requiring administration within 8 hours of APAP overdose to effectively block liver damage and achieve clinical treatment. Because early APAP overdose poisoning lacks specific clinical symptoms, patients often fail to detect the condition in time. Most patients who seek medical attention miss the optimal 8-hour treatment window and enter the late stage of liver damage. At this point, the efficacy of NAC treatment alone is significantly reduced, and the risk of liver failure and death is significantly increased.

[0005] In summary, existing clinical treatment protocols suffer from the core pain points of limited treatment time window and ineffective late-stage treatment. There is an urgent need to explore the downstream pathological mechanisms of AILI and develop novel combination drug regimens that can extend the treatment time window and are suitable for late-stage intervention in order to reduce the mortality rate of critically ill patients and meet the needs of clinical emergency care. Summary of the Invention

[0006] This invention aims to address the shortcomings of existing N-acetylcysteine ​​treatments for acetaminophen-induced acute liver injury (AILI), which have a narrow effective treatment window and are ineffective in late interventions. It provides a synergistic pharmaceutical composition and its application to achieve effective late intervention for AILI, extend the clinical treatment window, and reduce patient mortality.

[0007] To achieve the above objectives, the present invention employs the following technical means:

[0008] A pharmaceutical composition for treating acetaminophen-induced liver injury comprises a therapeutically effective amount of N-acetylcysteine ​​or a pharmaceutically acceptable salt thereof, and an 8-oxoguanine DNA glycosylation enzyme 1 specific activator. The pharmaceutical composition is a synergistic combination for late-stage intervention in acute liver injury caused by acetaminophen overdose. The two components, when used together, can jointly prolong the clinically effective therapeutic window of N-acetylcysteine, inhibit hepatocyte pyroptosis, and treat liver inflammation.

[0009] Preferably, the 8-oxoguanine DNA glycosylation enzyme 1 activator is TH10785 or a pharmaceutically acceptable derivative thereof.

[0010] Preferably, TH10785 or its pharmaceutically acceptable derivative is a functional synergist used to synergistically work with N-acetylcysteine ​​to prolong the effective therapeutic window of N-acetylcysteine ​​in treating acetaminophen-induced liver injury.

[0011] Preferably, the pharmaceutical composition can be prepared as an injection, an oral formulation, or a lyophilized powder for injection.

[0012] The use of a pharmaceutical composition, specifically the use of the pharmaceutical composition described above in the preparation of a medicament for treating acetaminophen-induced acute liver injury.

[0013] Preferably, the drug is used for late intervention treatment of acetaminophen intake beyond the conventional 8-hour therapeutic window of N-acetylcysteine.

[0014] Preferably, the drug achieves its therapeutic effect on liver injury through the following pathways: reducing the accumulation of 8-oxoguanine in the liver, inhibiting the formation of Z-DNA in hepatocytes, blocking the interaction between ZBP1 protein and Z-DNA, and inhibiting Caspase-8-mediated hepatocyte pyroptosis.

[0015] Preferably, the application is equipped with a treatment kit, which includes the pharmaceutical composition described above and an accompanying instruction manual, for the standardized implementation of the therapeutic application of the pharmaceutical composition.

[0016] The present invention has the following beneficial effects:

[0017] 1. Breaking through the limitations of traditional treatment time window: This invention, through the synergistic combination of NAC and TH10785, breaks through the clinical limitation that NAC is only effective for 8 hours, extending the effective intervention time of AILI to 12 hours and above, thus gaining valuable treatment time for poisoning patients who seek medical attention in the late stage and filling the gap in clinical late-stage treatment.

[0018] 2. Synergistic effect of dual targets, significant therapeutic effect: This invention adopts a dual-target treatment mechanism of "upstream detoxification + downstream damage control". NAC is responsible for supplementing GSH and neutralizing the early toxic metabolite NAPQI, while TH10785 is responsible for repairing DNA oxidative damage, blocking hepatocyte pyroptosis and inflammatory cascade reaction. The synergistic effect of the two can significantly reduce liver pathological damage, reduce transaminase levels, inhibit inflammatory infiltration, greatly improve the 96-hour survival rate of severely ill mice, and reduce clinical mortality compared with monotherapy.

[0019] 3. Innovative discovery of novel therapeutic targets: This invention is the first to apply the DNA oxidative damage repair, BZ DNA conformation regulation and ZBP1 innate immune pathway intervention strategy to the treatment of AILI, opening up a new direction for downstream targeted therapy of AILI. The target is novel and the mechanism is clear, with extremely high basic research value and clinical translation prospects.

[0020] 4. Wide dosage form adaptability and strong clinical applicability: The drug composition of this invention can be prepared as injection, oral preparation, and lyophilized powder injection, which is suitable for various clinical scenarios such as emergency care and inpatient treatment. The matching kit can realize standardized operation and facilitate clinical promotion and application. Attached Figure Description

[0021] Figure 1 A schematic diagram showing the levels of 8-oxoguanine (8-oxoG) in the livers of AILI mice in the TH10785 group and the control group;

[0022] Figure 2 This is a schematic diagram showing the levels of Z-NA in the livers of AILI mice in the TH10785 group and the control group.

[0023] Figure 3 A schematic diagram showing the interaction between ZBP1 and Z-NA in the livers of AILI mice in the TH10785 group and the control group;

[0024] Figure 4 A schematic diagram of pyroptosis-related proteins in the livers of AILI mice in the TH10785 group and the control group.

[0025] Figure 5 This is a schematic diagram showing the pathological analysis (H&E staining) of liver tissue and the results of TUNEL, CC3 and MPO immunofluorescence staining in AILI mice of the TH10785 group and the control group.

[0026] Figure 6 This is a schematic diagram showing the mRNA expression levels of various pro-inflammatory cytokines and chemokines (Tnf, Il1a, Il1b, Il6, Cxcl1, Cxcl2, Ccl2) in the livers of TH10785 group and control AILI mice.

[0027] Figure 7 This is a schematic diagram showing the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in AILI mice of the TH10785 group and the control group.

[0028] Figure 8 A schematic diagram showing the 96-hour survival rate of AILI mice treated with NAC and TH10785 alone and in combination. Detailed Implementation

[0029] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] This invention provides a pharmaceutical composition for treating acetaminophen-induced liver injury, comprising a therapeutically effective amount of N-acetylcysteine ​​or a pharmaceutically acceptable salt thereof, and an 8-oxoguanine DNA glycosylation enzyme 1 specific activator. The two components work synergistically and are specifically designed for late-stage intervention in the treatment of acute liver injury caused by acetaminophen overdose.

[0031] The 8-oxoguanine DNA glycosylation enzyme 1 activator is preferably TH10785 or a pharmaceutically acceptable derivative thereof. This component, as a functional synergist, can synergistically work with N-acetylcysteine ​​to significantly prolong its clinically effective therapeutic window.

[0032] The pharmaceutical compositions of the present invention can be prepared as injections, oral formulations or lyophilized powder injections by adding pharmaceutically acceptable carriers, diluents or excipients according to conventional pharmaceutical processes, to suit different clinical administration scenarios.

[0033] The present invention also provides the use of the pharmaceutical composition, specifically its use in the preparation of a drug for treating acetaminophen-induced acute liver injury.

[0034] The term "drug" specifically refers to a late-stage intervention treatment for acetaminophen intake exceeding the traditional 8-hour therapeutic window of N-acetylcysteine.

[0035] The therapeutic mechanism of the pharmaceutical composition of the present invention is as follows: by activating 8-oxoguanine DNA glycosylation enzyme 1, the accumulation of 8-oxoguanine in the liver is reduced, the DNA BZ conformational transition in hepatocytes is inhibited, and Z-DNA generation is blocked, thereby blocking the specific interaction between ZBP1 protein and Z-DNA, inhibiting the Caspase-8-mediated hepatocyte pyroptosis pathway, and at the same time reducing local inflammatory infiltration in the liver, thus blocking the downstream liver injury cascade reaction.

[0036] This invention provides a treatment kit containing the pharmaceutical composition of this invention and a standardized instruction manual, enabling standardized and regulated clinical application in treatment.

[0037] Example 1 (Regulatory effects of TH10785 on oxidative damage, pyroptosis and inflammation in the liver of AILI mice)

[0038] 1. Establishment of experimental animals and models

[0039] Eight-week-old male wild-type (WT) mice were selected, and after acclimatization, they were fasted for 16 hours and then injected intraperitoneally with 300 mg / kg acetaminophen (APAP) to establish a mouse model of acute liver injury.

[0040] 2. Experimental grouping and drug intervention

[0041] One hour after APAP modeling, the mice were randomly divided into two groups:

[0042] Control group: Intraperitoneal injection of an equal volume of solvent;

[0043] TH10785 group: TH10785 was administered intraperitoneally at a dose of 20 mg / kg.

[0044] 3. Detection Indicators and Methods

[0045] Six hours after drug intervention, mice were sacrificed, liver tissue was dissected, and serum samples were collected for multiple assays: ELISA was used to detect liver 8-oxoG content to assess the degree of DNA oxidative damage; Z22 antibody immunofluorescence staining was used to detect hepatocyte Z-DNA levels; PLA (partial linkage) technique was used to detect the interaction between ZBP1 protein and Z-DNA; Western blotting was used to detect the expression levels of hepatocyte pyroptosis-related proteins; H&E staining was used for liver histopathological analysis, combined with TUNEL, CC3, and MPO immunofluorescence staining to detect liver damage area, hepatocyte death, apoptosis, and neutrophil infiltration; qRT-PCR was used to detect the expression levels of hepatic pro-inflammatory cytokines and chemokine mRNA; and serum biochemical assays were used to measure ALT and AST levels to assess the degree of liver damage.

[0046] 4. Experimental Results

[0047] Combination Figure 1-7 The results showed that, compared with the control group, the TH10785 treatment group had significantly reduced 8-oxoG accumulation in the liver, significantly reduced pathological Z-DNA production, and significantly inhibited the interaction between ZBP1 and Z-DNA. Simultaneously, the expression of hepatocyte pyroptosis-related proteins was downregulated, and the area of ​​liver tissue necrosis, hepatocyte mortality, and neutrophil infiltration were significantly reduced. The expression of various pro-inflammatory factors such as Tnf, Il1a, Il1b, and Il6 was significantly decreased, and serum ALT and AST liver function indicators were significantly improved. This demonstrates that TH10785 can effectively repair APAP-induced liver DNA oxidative damage, block the Z-DNA / ZBP1-mediated hepatocyte pyroptosis pathway, inhibit the liver inflammatory cascade, and alleviate acute liver injury.

[0048] Example 2 (Effect of combined drug administration on the survival rate of mice with advanced AILI)

[0049] 1. Establishment of experimental animals and lethal models

[0050] Eight-week-old male C57BL / 6 wild-type mice were selected, and after fasting for 16 hours, they were injected intraperitoneally with 600 mg / kg APAP to construct a lethal acute liver injury model, simulating a late-stage case of severe drug poisoning in clinical practice.

[0051] 2. Experimental grouping and late-stage drug intervention

[0052] Twelve hours after APAP modeling (exceeding the traditional 8-hour treatment window of NAC), mice were randomly divided into four groups for late-stage intervention treatment:

[0053] Control group: Intraperitoneal injection of PBS solvent;

[0054] NAC monotherapy group: 100 mg / kg NAC administered intraperitoneally;

[0055] TH10785 monotherapy group: 20 mg / kg TH10785 administered intraperitoneally;

[0056] Combination therapy group: intraperitoneal injection of 100 mg / kg NAC + 20 mg / kg TH10785.

[0057] 3. Observation indicators

[0058] The mice were observed continuously for 96 hours, and the number of surviving mice in each group was recorded daily, and the survival rate was calculated.

[0059] 4. Experimental Results

[0060] like Figure 8As shown, under late intervention conditions of 12 hours after APAP poisoning, the NAC monotherapy group only had a weak protective effect, while the TH10785 monotherapy group had a protective effect comparable to that of the late-stage NAC monotherapy group. However, the 96-hour survival rate of mice treated with the combination of NAC and TH10785 was significantly higher than that of all monotherapy groups and the control group. This confirms that the combination of the two drugs can achieve a significant synergistic effect, effectively breaking through the limitations of traditional treatment time windows, significantly improving the prognosis of mice with late-stage severe APAP liver injury, and reducing mortality.

[0061] Example 3 (Preparation process of the pharmaceutical composition)

[0062] 1. Preparation of injection: Take a therapeutically effective amount of N-acetylcysteine ​​and TH10785, dissolve them together in sterile physiological saline or phosphate buffer, stir thoroughly to dissolve, adjust the pH of the solution to 7.0-7.4, filter through a sterile filter membrane to remove bacteria, and aseptically dispense to obtain the finished injection.

[0063] 2. Preparation of lyophilized powder for injection: The sterile drug solution prepared above is pre-frozen, vacuum freeze-dried, and sealed by compression to prepare lyophilized powder for injection, which is convenient for long-term storage and clinical transportation. It can be reconstituted with sterile solvent before use.

[0064] 3. Preparation of oral formulations: Following conventional pharmaceutical processes, N-acetylcysteine, TH10785, and pharmaceutically acceptable excipients such as starch, lactose, and microcrystalline cellulose are mixed evenly, granulated, dried, tableted, or filled into capsules to produce oral formulations such as tablets and capsules.

[0065] Example 4 (Preparation of Standardized Treatment Kit)

[0066] The accompanying treatment kit of this invention includes the NAC+TH10785 drug composition preparation described above, as well as a standardized clinical instruction manual. The instruction manual clearly indicates the standardized operating procedures such as dosage, administration time, applicable population, indications for late intervention, and administration method, which can realize the standardized and regulated application of clinical AILI late-stage treatment.

[0067] The examples provided in this invention are not intended to limit the implementation. Those skilled in the art will recognize that various variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations, and any obvious variations or modifications derived therefrom are still within the scope of this invention.

Claims

1. A pharmaceutical composition for treating acetaminophen-induced liver injury, characterized in that, The drug composition comprises a therapeutically effective amount of N-acetylcysteine ​​or a pharmaceutically acceptable salt thereof, and an 8-oxoguanine DNA glycosylation enzyme 1 specific activator. This pharmaceutical composition is a synergistic combination for late-stage intervention in acute liver injury caused by acetaminophen overdose. The two components, when used together, can jointly prolong the clinically effective therapeutic window of N-acetylcysteine, inhibit hepatocyte pyroptosis, and treat liver inflammation.

2. The pharmaceutical composition for treating acetaminophen-induced liver injury according to claim 1, characterized in that, The 8-oxoguanine DNA glycosylation enzyme 1 activator is TH10785 or a pharmaceutically acceptable derivative thereof.

3. The pharmaceutical composition for treating acetaminophen-induced liver injury according to claim 2, characterized in that, The TH10785 or its pharmaceutically acceptable derivatives are functional synergistic components used to synergistically work with N-acetylcysteine ​​to prolong the effective therapeutic window of N-acetylcysteine ​​in treating acetaminophen-induced liver injury.

4. The pharmaceutical composition for treating acetaminophen-induced liver injury according to claim 1, characterized in that, The pharmaceutical composition can be prepared as an injection, an oral formulation, or a lyophilized powder for injection.

5. The application of a pharmaceutical composition, characterized in that, The use of the pharmaceutical composition according to any one of claims 1-4 in the preparation of a pharmaceutical composition for treating acetaminophen-induced liver injury.

6. The application of the pharmaceutical composition according to claim 5, characterized in that, The drug is used for late-stage intervention treatment of acetaminophen intake beyond the conventional 8-hour therapeutic window of N-acetylcysteine.

7. The application of the pharmaceutical composition according to claim 5, characterized in that, The drug achieves its therapeutic effect on liver injury through the following pathways: reducing the accumulation of 8-oxoguanine in the liver, inhibiting the formation of Z-DNA in hepatocytes, blocking the interaction between ZBP1 protein and Z-DNA, and inhibiting Caspase-8-mediated hepatocyte pyroptosis.

8. The application of the pharmaceutical composition according to claim 5, characterized in that, The application is equipped with a treatment kit, which includes the pharmaceutical composition according to any one of claims 1-4 and an accompanying instruction manual, for the standardized implementation of the therapeutic application of the pharmaceutical composition.

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